extfieldsofstudy: Biology Medicine

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Glyco-biomarkers: Potential Determinants of Cellular Physiology and Pathology

Once dismissed as just the icing on the cake, sugar molecules are emerging as vital components in life’s intricate machinery. Our understanding of their function within the context of the proteins and lipids to which they are attached has matured rapidly, and with it the far reaching clinical implications are becoming understood. Recent advances in high-throughput glycomic techniques, glyco biomarker profiling, glyco-bioinformatics and development of increasingly sophisticated glyco-arrays, combined with our increased understanding of the molecular details of glycosylation have facilitated the linkage between aberrant glycosylation and human diseases, and highlighted the possibility of using glyco-biomarkers as potential determinants of disease and its progression. The focus of this review is to give an insight into the biological significance of these glycomodifications, highlight some specific examples of glyco-biomarkers in relation to autoimmunity and in particular rheumatoid arthritis, and to explore the exciting possibility of exploiting these for diagnostic and prognostic strategies.

Introduction
The recent surge of interest in glycomics and the search for glyco-biomarkers of disease stems from the fact that glycosylation has, finally, gained recognition for the pivotal role that it plays in virtually all aspects of our system; from embryogenesis to pathogenesis [1][2][3][4][5][6]. This comes as no surprise, since the surface of our entire cellular network, as well as those of pathogens, and the backbone of most proteins and lipids, is decorated with a dense complement of either linear or intricately branched complex sugar structures [3,5,7].
These glycans constitute the most abundant and diverse of the post-translation modifications in our sys-tem and are therefore an integral feature of almost all biomolecules including nearly all cell surface and over 70% of secretory proteins, as well as glycolipids, glycosphingolipids (such as the ABO histo-blood group antigens), lipopolysaccharides, and glycosaminoglycans (GAGs; such as heparin). In the majority of these examples the glycan moiety constitutes a substantial portion of the mass, size and charge of the glycoconjugate, and can thus exert considerable inter-and intramolecular effects. As such glycans have the potential to generate extensive physical and biochemical diversity (through the formation of large numbers of glycoforms), and therefore confer considerable coding capacity for relay of biospecific information. And so, in addition to performing a structural and protective role, a large number of glycans have important functional roles as specific information tags or recognition epitopes [3,8].
The information coded into these, spatially accessible, sugar epitopes is decoded by a sophisticated recognition system, which is comprised of a large cohort of carbohydrate binding proteins that include lectins, Table 1 A list of some of the endogenous lectins/carbohydrate-recognizing proteins expressed in our system. Depending on their structure and mode of action, lectins are subdivided in several groups. These lectins contain one or more carbohydrate recognition domains that determine their specificity.

Endogenous lectins Function Annexins
Functions include binding to carbohydrate moieties of sialoglycoproteins and GAGs. Examples include annexin IV, in kidney and pancreas. May be important apical sorting (secretory vesicles) and exocrine-type neurotrophic activity and in cell-adhesion (or inhibition of cell-adhesion).
C-type lectins: calciumdependent Soluble/ transmembrane Collectins; soluble lectins such as C-reactive protein (CRP), mannose binding lectin (MBL), surfactants SP-A and SP-D and ficolins, which have the capacity to activate complement and thus play an important role in innate immunity as well as autoimmunity Selectins; membrane bound with specific function in leukocyte adhesion to endothelial cells through sialyl-LewisX recognition; therefore important in both normal physiology and in inflammation and immunity to tumor and virally infected cells. A rapidly growing family of metal-independent lectins with diverse histological localization, in cytoplasm, nuclei, cell surfaces and extracellular spaces; depending on the galectin species. They share galactose-specificity and display potent biological activities, such as the ability to induce apoptosis, or metabolic changes, such as cellular activation and mitosis. Examples include galectin 1 which induces T cell apoptosis and galectin 3, associated with tumours, which inhibits apoptosis collectins, adhesion molecules, and anti-carbohydrate antibodies (Table 1) [8][9][10][11][12]. This versatile carbohydrate recognition system combined with our extensive glycome are key players in orchestrating the complex functional network of bimolecular interactions that coordinate molecular and cellular function in relation to innate and adaptive immunity [13][14][15][16][17][18][19]. Given the diversity of structures and functions, and the potential for conveying information essential to maintenance of immune homeostasis, it is not surprising that the role of glycosylation in the development, regulation, and progression of disease has come under increased scrutiny [6].

Physiological diversity and function of glycans
As might be imagined from their ubiquitous nature, and their ability to convey information, the biological roles of glycans are formidable and span the complete spectrum, from those that are relatively subtle (e.g. structural) to those that are critical (e.g. crucial for the development, function and survival of an organism).
However, the elucidation of a specific physiological role for a given glycan modification(s) poses a formidable challenge. This is because glycan structures can play different roles in different cells/tissues, at different times [3,[20][21][22], and also because in some instances what is deemed structural under normal physiological conditions may be rendered antigenic in certain disease conditions. Examples of these include 1) the developmentally regulated expression and distribution of the ABO histo-blood group antigens [23], and 2) the glycosylation changes that render certain collagen epitopes arthrogenic [24].
The plethora of biological functions ascribed to glycans include mechanisms of protein folding and turnover [25], trafficking and distribution [26,27], pharmacokinetics [28], as well as immunogenicity; where glycomodifications may lead to unmasking of antigenic epitopes in the glyconcjugate backbone (e.g. antigenic peptide sequences) or reveal/create glyco-antigenic determinants (through aberrant exposure of certain terminal sugar residues e.g. N -acetylglucosamine, or via aberrant changes to the core structure e.g. unusual chain elongation/branching) [29,30].
The latter may be of particular relevance as it adds credence to the emerging, sometimes controversial, linkage between blood group antigen expression and disease [41][42][43][44][45][46], in which certain ABO antigens are implicated because of their role as microbial glycan receptors, as tumor antigens and as ligands for important immunologic reactions [5,47,48].
Glycans are also important components of cytosolic and nuclear proteins. An important example of this is the dynamic modification of these proteins with Olinked β-N -acetylglucosamine (O-GlcNAc). The covalent attachment of this O-GlcNAc (to serine or threonine residues) has been shown to be a regulatory posttranslational modification that is responsive to various stimuli. Its main function is that of a regulatory switch in the metabolic control of signal transduction, transcription, stress response, apoptosis, as well as T-and B-lymphocytes activation [17,49]. O-GlcNAc glycosylation is also critical in both neuronal function and dysfunction (neuronal signaling and synaptic plasticity) and may have a crucial impact on the nervous system and consequently various neurodegenerative diseases [50].
An appreciation of the importance of glycans and their multidimensional roles in various physiological and pathological circumstances would be incomplete without some degree of insight into their biosynthesis and the factors that determine their structural complexity and diversity, as discussed below.

Determinants of cellular physiology/pathology
A unique feature of glycosylation is the fact that despite its complexity and precise nature the biosynthesis of glycans can not be directly predicted from the DNA template [ Fig. 1], but is instead governed by an elaborate mechanism that utilizes a multitude of glycoenzymes [21]. These enzymes, which display exquisite biological specificity, are expressed in a cell/tissuespecific, and temporally regulated manner. Their expression is controlled by multiple tissue-specific promoters that may be activated/suppressed under different physiological circumstances (e.g. expressed at discrete points in lymphocyte development and peripheral activation) [35]. Examples of this include the way in which pro-inflammatory or anti-inflammatory cytokines alter the expression of specific glycosyltransferases; that in turn regulate the conversion of activated T cells into memory cells, or the differentiation of Th cells into Th1 and Th2 subsets and thereby influence disease outcome [18,51].
It is therefore not surprising that the glyco-profile of a given cell/tissue (including serum) can alter in response to a whole host of physiological [40,52,53] or pathological situations e.g. angiogenesis, immune challenge, inflammation or oncogenic transformation and metastasis [3,8,11,54].
An understanding of this code as it relates to disease states, at both molecular and functional levels can help unravel disease mechanisms and thus pathology.

Glycosylation and disease
In accord with the above observations, aberrant changes in cellular processes, such as those that accompany disease, are therefore likely to result in alterations of the glycan profiles of the cell surface and/or secreted glycoconjugates, in particular glycoproteins. And so, not surprisingly, most major diseases, when probed, are found to be directly/indirectly associated with a change in the glycosylation pattern of at least one central structure.
The glycosylation changes in relation to these and many other diseases, not covered here, range from the subtle to the palpable and can be acquired/inherited.

Inherited glycosylation diseases; rare or under diagnosed?
Given the critical role of glycosylation, all the inherited glycosylation diseases detected so far are autosomal recessive disorders that are due to polymorphisms and minor mutations and never the result of gross mutations (which are unlikely to occur as they would be fatal at the early stages of fertilization/embryogenesis). Those that occur are relatively rare, and give rise to either severe phenotypic consequences (sometimes with neonatal death) or very minor ones; which are likely to be asymptomatic. The underdiagnosis of this group of diseases is further compounded by ascertainment bias, due to pleiotropic or unpredictable phenotypes.
The plethora of different mutations detected results in a diverse group of diseases including leukocyte adhesion deficiency syndrome II, congenital dyserythropoietic anemia type II (also known as HEMPAS), and an emerging varied group of disorders (> 20 separate genes and more than 100 allelic variants since the first report in 1980, the majority of which were identified in the past few years) collectively known as congenital disorders of glycosylation (CDGs) [72].
By far the most frequent of these is CDG-Ia. This group of diseases (> 60 mutations in phosphomannomutase-2) have a variable clinical spectrum, ranging from disorders restricted to specific organs to severe multisystemic disorders, including central nervous system phenotypes [72], suggesting that the brain is particularly susceptible to perturbations in glycosylation.
As these glycosylation disorders are likely to target and affect a large set of proteins/lipids, it is not hard to see how glycosylation enzymes might act as modifiers of other gene defects and thus modulate the severity and nature of diseases such as various neuropsychiatric conditions including Alzheimers and Schizophrenia [61].
The diseases discovered so far, however, may represent only the "tip of the iceberg" [72,73], as it is estimated that defects in any of the well over 50-100, critical glycosylation genes will cause diseases such as those described above, and many of these can only be detectable by sugar profiling [74].

Acquired glycosylation diseases
Aberrant, non-inherited, glycomodifications are extensive and invariably found to be associated with, or a pre-requisite for, a wide-ranging myriad of disease phenotype [2][3][4]6].
The most obvious of these are changes that accompany angiogenesis or disease associated immune processes such as cellular activation, recruitment and inflammation; all of which encompass sugar changes of one type/another e.g. glycomodification of various activation/co-stimultory cell surface molecules on cytotoxic/helper T cells, as well as various ligands such as sLex [40], which act as tissue specific zip codes regulating lymphocyte traffic to a given site/organ [3,75,76].
However, in addition to these, there is also a catalogue of "disease specific glycosylation changes" that play a key role in the actual disease mechanism, and are thus of pathophysiological significance [3,33,58,77,78].
These fall into two categories: In some instances, as for example cancer, altered glycosylation is a universal feature that reflects significant changes in certain specific glyco-enzymatic pathways and thus closely correlates with critical aspects of the disease, whilst in others the glycomodifications may be more subtle, acting as a trigger that could instigate biological effects that may initiate or, in certain circumstances, alter the course of disease.
The impact of these glycosylation changes in relation to autoimmunity and in particular RA pathology will be the main focus of this section, followed by a brief overview of a few select examples of other autoimmune diseases/disease mechanisms where specific glycosylation changes play a critical role.

Glyco-biomarkers and autoimmunity
Glycomodification may represent one way in which immune tolerance can be bypassed. Some posttranslational modifications can create new self antigens (Ags) or even mask Ags normally recognized by the immune system [24,30,[79][80][81][82]. The former is of particular relevance since even subtle changes may lead to immune activation resulting from recognition of the glyco-Ags by the naturally occurring anti-glycan auto antibodies (AutoAbs); directed against a vast repertoire of non-self glycan structures [83] existing on bacterial, fungal and parasite cells.
As such the link between glycomodifications and autoimmunity is complex and includes: (i) Cross-reactivity: Unlike classical peptide epitopes, glyco epitopes (glycotopes) can share significant structural homologies. As such they can display extensive cross-reactivity and thus behave as "panepitopes", which may, in some contexts, be implicated in autoimmunity. A classic example of this is the possible recognition of abnormally exposed N -Acetylglucosamine (GlcNAc) in our system (resulting from aberrant hypogalactosylation; as seen in RA IgG) by pathogen associated molecular pattern receptors of innate immunity such as mannose binding lectin (MBL), which may result in immune dysregulation [9]. (ii) Neo-expression: Expression of glycans normally restricted to other tissues or molecules [84] or incorporation of immunogenic non-human dietary glycans e.g. N -Glycolyneuraminic acid [85], which could generate xenoreactive, and potentially autoreactive Ab responses and cause long-term inflammatory reactions.
(iii) Inappropriate processing and presentation: In some instances glycan changes can lead to altered processing and presentation of selfantigens to T cells via major histocompatibility complex (MHC). An interesting example of this is the role of collagen derived remnant glycotopes in autoimmune arthritis [24]. The latter has been the focus of extensive studies as will be discussed below.

IgG glyco-biomarkers and rheumatoid arthritis
The N -linked glycans at the single, highly conserved, glycosylation site in the constant domain of IgG Fc region (which can be present in any one of 30 variations depending on the presence/absence of absence of galactose, sialic acid, bisecting N -acetylglucosamine [bis-GlcNAc] and fucose; Fig. 2) are critical features, which have far reaching structural and functional impact. Affecting both the innate and adaptive arms of the immune response [33,87,88].
During the past two decades particular emphasis has been placed on the aberrant glycosylation of IgG and its role in RA pathogenesis [3,57,58,[89][90][91][92][93][94]. The research in this field has been extensive and has drawn together all aspects of aberrant IgG glycosylation, including the structural anatomy of the IgG, the clinical implications in both human disease and animal models, the glycosylation enzymes and the genes that encode and regulate these enzymes, and the possible pathogenic mechanisms of glyco-modified IgG [3,21,24,57,58,86].
The enormous volume of data generated suggests that RA may be a dysregulated glycosylation disease in which IgG glycomodifications may play a pivotal role. These glycomodifications have been shown to be far more complex than previously thought, encompassing not only galactosylation, but also fucosylation and sialylation, as well as the additional microheterogeneity of both symmetrical and asymmetrical pairing of the Fc glycans [3,58,95].
The loss of galactose and the increased levels of core fucosylation affect the interface between IgG-Fc fragments and its interaction with other molecules and consequently interfere with Fc receptor binding and effector activities [88] causing defective/altered effector/regulatory mechanisms of the immune and thus inflammatory response [3,24,58].

IgG Glyco-biomarkers as useful diagnostic
tools for RA The rheumatic diseases are heterogeneous (Fig. 3), and overlapping disease syndromes may be found [57]. At present there is no single diagnostic test capable of differentiating one disease from another. Increased levels of IgG-G0 have proven to be useful biomarkers of RA. Their presence in serum predates the onset of RA by at least 10 years, and in early synovitis has been found to be associated with the development of RA; such that when combined with RF it has 90% sensitivity, 95% specificity and 94% positive predictive value [3]. This has prompted the use of sugar printing for the differentiation of rheumatic diseases [55][56][57]. Sugar printing of serum IgG can differentiate early rheumatoid arthritis (ERA) and RA from each other and from other rheumatic diseases and hence may constitute a relatively rapid diagnostic test for patients presenting with arthritis [57]. In the case of RA, it was found that ERA/RA are distinguished from other diseases by their IgG-G0 and -G0F (the most abundant G0 structure in RA), but differ from each other by their monogalactosyl (G1) and sialylated sugar profiles. Undifferentiated arthritis, in contrast, had no specific distinguishing features, as one would expect from what is probably a heterogeneous group of pathologies. The strength of the association with RA is confirmed by the fact that G0 and G0F can be used to predict RA from the pool of patients with a broad spectrum of disease. Although the study was not designed for the purpose of evaluating the diagnostic utility of IgG-G0/-G0F per-se, over 75% of RA patients were correctly identified as having the disease and the test was shown to have a sensitivity of 50%, and a specificity of 84% [57].
Structural studies on IgG oligosaccharides have also proved useful in unraveling some of the clinical overlap in patients with primary Sjogren's syndrome (SS); whereby the appearance of IgG-G0 in primary SS may be related to future complication with RA [96].
Clinical studies in this field strongly support the proposed relationship between IgG glycosylation, immune complex formation, increased rheumatoid factor (RF) avidity, as well as MBL binding and pathology in RA. They demonstrate that increased IgG-G0 (circulating in the serum and/or in immune complexes in the synovial fluid), correlate with increased RF avidity, with higher tender joint score, and a higher frequency and number of subcutaneous nodules in RA patients [90,[97][98][99].

IgG Glyco-biomarkers as useful prognostic tools for RA
In addition, IgG-G0 has also proved to be a good prognostic indicator of RA. High incidence of serum IgG-G0 is related to disease activity and the progression to erosive articular changes, as determined in a well-characterized cohort of 127 female RA patients who were followed for a mean duration of 6 years. IgG-G0 values correlated with the number of erosions, disease activity and served as an indicator for disease progression [3,57,58,98].
IgG-G0 not only correlates with severity and duration of disease [90], but has also been shown to return to normal levels following treatment e.g. with anti-TNF [100]. The latter observation is in-line with changes observed in pregnancy where the decrease in IgG-G0 levels is associated with a remission in the disease, and where a rapid rebound increase in IgG-G0, post partum, is associated with disease flares in RA patients [91]. The latter is of particular importance as it further supports the notion that IgG-G0 may be a susceptibility factor in the development of RA.
In this respect IgG glyco-biomarkers may be very useful in determining the clinical efficacy of im-munotherapeutic agents such as the new biologics [100], which are revolutionizing the way that we treat autoimmune diseases such as RA and SLE [3].

IgG glyco-biomarkers in other rheumatological diseases
The appearance of IgG-G0 is also a general feature of other unrelated chronic granulomatous diseases e.g. Crohn's disease (CD) and Mycobacterium tuberculosis, as well as a restricted group of other rheumatological diseases such as SLE, SS, psoriatic arthritis and juvenile idiopathic arthritis (JIA) [56]. However, detailed sugar profiling of these diseases has demonstrated differential patterns of Fc-glycomodifications; encompassing Gal, Fuc, Bis-GlcNAc and sialic acid; and supports the notion that each disease may be associated with a distinct pattern of IgG glycosylation [3,[55][56][57][58].
Glycosylation-related pathology is not unique to IgG, and can include other immunoglobulins (e.g. IgA) and other immunologically pertinent molecules such as the acute phase proteins, as well as mucins, as summarized below.

IgA glyco-biomarkers in IgAN and
Henoch-Schönlein Purpura (HSP) IgAN is defined by the deposition of IgA1 in the glomerular mesangium, whilst HSP is a form of systemic vasculitis characterized by tissue deposition of IgA. Nephritis with IgA1 deposition is a common feature of HSP and histologically indistinguishable from IgAN. Analysis of serum IgA1 from patients with the IgA nephropathy showed decreased galactosylation (and hence sialylation) of the O-glycans in the hinge region of IgA1 (IgA1 has 5 sets of closely located Oglycans in this region). This hypogalactosylation corresponds to decreased B cell β1 3-galactosyltransferase activity, which may be altered due to increased production of Th2 cytokines [66,101].
Altered glycosylation of IgA1 results in a loss of conformational rigidity, which may explain the increased formation of aggregates, and the glomerular accumulation of IgA1 [66].

Glyco-biomarkers in Inflammatory Bowel
Disease (IBD) IBD is a chronic intestinal disorder comprising two major types: ulcerative colitis (UC) and CD; with considerable overlap. The diagnosis and the differentiation between UC and CD is therefore invariably dependent on a combination of clinical, serological, endoscopic, histopathologic and radiological characteristics; with > 15% of patients being diagnosed with indeterminate colitis. The degree of glycosylation of the mucins is central to their role in IBD. It has been demonstrated that the degree of sulphation and sialylation and the length of the oligosaccharide chains can vary, and thus affect the function of mucins as a protective barrier. The changes are different in UC compared to CD. In UC, the sialic acids of the colonic mucosa, which are normally heavily O-acetylated, lose this modification [102]. This may have pathogenic significance as these modifications do render the sialic acids more resistant to bacterial sialidases.

Anti-glycan Auto-antibodies (AutoAbs) as biomarkers of disease
As already pointed out glycan structures share significant structural homologies and can act as cross reactive panepitopes. These may in certain circumstances be highly antigenic and instigate the activation and increased production of naturally occurring autoAbs, which may result in inflammation and autoimmunity.

Auto-abs against IgG-G0 as biomarkers in RA
and JIA The increased IgG-G0 in RA has been shown to be associated with presence of increased levels of anti IgG-G0 auto-Abs. Studies examining the diagnostic value of anti IgG-G0 auto-Abs, in 266 Japanese patients with systemic autoimmune diseases, including 60 with RA, suggests that anti IgG-G0 Abs may be a more specific marker for RA than conventional IgM RF [97], which is the current biomarker of choice for the diagnosis of RA.
These anti IgG-G0 autoAbs, show a significant correlation with C reactive protein levels and have a higher sensitivity in detecting immunological disorders in JIA and juvenile onset Sjogren's syndrome when compared with RF [97,103].

Auto-abs against neuronal glycans in certain
neuropathies A number of neuropathies are associated with circulating auto-Abs directed against certain glycan epitopes that are highly expressed in the nervous system (e.g. the sugar chains of gangliosides), resulting in autoimmune nerve damage [78]. Anti-ganglioside IgM Abs can cause leakage of the blood-nerve barrier in a concentration-dependent and complementindependent manner, and can also bind to neuronal gangliosides (to create a neuromuscular block) and serve as a marker of axonal damage in neuropathies such as multiple sclerosis [104].
The majority of these auto-Abs originate either from our naturally occurring pool of anti-glycan Abs (B cell clones; usually germ-line encoded), or are the result of exposure to bacterial antigens. Examples include: i) Monoclonal IgM or IgA Abs; highly specific for either ganglio-series gangliosides, or sulfated glucuronosyl glycans (the so-called HNK-1 epitope), secreted by benign or malignant B-cell neoplasms [105]. The presence of these Abs is associated with the onset of symptoms of a demyelinating neuropathy involving the peripheral and central nervous systems: the Guillain-Barre and Miller-Fisher syndromes, respectively. ii) Cross reactive AutoAbs; directed against gangliosides structures such as G M1 and G Q1b , which occur following infection with bacteria such as Campylobacter jejuni, which mimic ganglioside structures [106].

Auto-Abs against Tn antigen in
Tn-Polyagglutinin Syndrome Anti-Tn Abs are yet another example of naturally occurring anti-glycan Abs present in our sera. Tn polyagglutinability syndrome is an acquired condition where the blood cells made by the bone marrow express the Tn antigen; O-linked N-acetylgalactosamine (GalNAcα1-O-Ser/Thr) and the sialylated-Tn (SA α2,6 GalNAcα1-O-Ser/Thr), thus becoming susceptible to hemagglutination by the naturally occurring anti-Tn Abs [107]. This change appears to be due to acquired stem-cellbased loss of expression of the O-glycan Core 1 β1-3 galactosyltransferase activity, which occurs despite the fact that there may be as many as at least five distinct gene loci encoding additional copies of this enzyme. Patients with this syndrome show a wide range of symptoms. Some have varying degrees of hemolytic anemia and/or decreases in other blood cell types, whilst others have no detectable symptoms and are only picked up through blood typing. Although the mechanisms are unclear, the presence of this syndrome is, in some patients, associated with an increased risk of developing subsequent leukemia.

Future developments
Recent developments in the use of glycan arrays for systematic screening of blood samples has lead to the discovery of a panel of anti-glycan antibodies which may prove useful as biomarkers enabling better diagnosis and prognosis of diseases such as Crohn's disease and multiple sclerosis (MS).

Anti-Glycan Abs as biomarkers for CD
One of the major serological markers for CD is anti-Sacharomyces cerevisiae Abs, which is directed against oligomannosidic residues on the polysaccharide mannan in the cell walls of the yeast S. cerevisiae. Recent systematic screening for anti-glycan antibodies in CD using glycan array have lead to the discovery of novel anti-glycan abs [83]. These include anti-laminaribioside (Glc(β1,3)Glc(β)) and antimannobioside (Man(α1,3)Man(α)) glycan IgG Abs, as well as anti-chitobioside (GlcNAc(β 1,4)GlcNAc(β)) glycan IgA Abs; with the latter demonstrating the highest discriminative capability between CD and UC. Combination of these anti-glycan biomarkers have been shown to be useful prognostic tool; predicting severe and complicated CD (presence of strictures or fistulas) and the need for surgical intervention.

Anti-Glycan Abs as biomarkers for diagnosis of
MS MS is an inflammatory demyelinating disease of the central nervous system. The disease is autoimmune in nature and is driven by a primary T-celldriven aberrant immune response, as well as an antigen driven B-cell responses. The panel of antibodies include a notable number of anti glycan antibodies, anti-galactocerebroside IgG and anti-Glc(α1,4)Glc(α) IgM auto-Abs. The levels of the latter were found to be significantly elevated in MS patients in comparison to other neurological diseases (with a 57% sensitivity and 85% specificity) [83]. These anti-glyco auto-Abs maybe particularly useful for the early diagnosis and prognosis of the relapsing -remitting form of MS.

Conclusion
There is a pressing need to develop new biomarkers that will serve as more sensitive diagnostic and prognostic tools which could be used to discriminate between different forms/stages of disease and to monitor the efficacy of various new treatment options. Glycobiomarkers have the potential to fulfill this need by providing better link between specific mRNAs, their corresponding polypeptides, glycoforms and cell function, and may thus provide a better insight into cellular and molecular interactions and therefore disease mechanisms; promising a new era in the interpretation of data relevant to immunotherapy and the design of new oligosaccharide-based diagnostics and therapeutics.


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Mouse Apolipoprotein B Editing Complex 3 (APOBEC3) Is Expressed in Germ Cells and Interacts with Dead-End (DND1)

Background The dead-end (Dnd1) gene is essential for maintaining the viability of germ cells. Inactivation of Dnd1 results in sterility and testicular tumors. The Dnd1 encoded protein, DND1, is able to bind to the 3′-untranslated region (UTR) of messenger RNAs (mRNAs) to displace micro-RNA (miRNA) interaction with mRNA. Thus, one function of DND1 is to prevent miRNA mediated repression of mRNA. We report that DND1 interacts specifically with APOBEC3. APOBEC3 is a multi-functional protein. It inhibits retroviral replication. In addition, recent studies show that APOBEC3 interacts with cellular RNA-binding proteins and to mRNA to inhibit miRNA-mediated repression of mRNA. Methodology/Principal Findings Here we show that DND1 specifically interacts with another cellular protein, APOBEC3. We present our data which shows that DND1 co-immunoprecipitates APOBEC3 from mammalian cells and also endogenous APOBEC3 from mouse gonads. Whether the two proteins interact directly remains to be elucidated. We show that both DND1 and APOBEC3 are expressed in germ cells and in the early gonads of mouse embryo. Expression of fluorescently-tagged DND1 and APOBEC3 indicate they localize to the cytoplasm and when DND1 and APOBEC3 are expressed together in cells, they sequester near peri-nuclear sites. Conclusions/Significance The 3′-UTR of mRNAs generally encode multiple miRNA binding sites as well as binding sites for a variety of RNA binding proteins. In light of our findings of DND1-APOBEC3 interaction and taking into consideration reports that DND1 and APOBEC3 bind to mRNA to inhibit miRNA mediated repression, our studies implicate a possible role of DND1-APOBEC3 interaction in modulating miRNA-mediated mRNA repression. The interaction of DND1 and APOBEC3 could be one mechanism for maintaining viability of germ cells and for preventing germ cell tumor development.

Introduction
The Dnd1 gene is essential for primordial germ cell survival in vertebrates from zebrafish, xenopus to mice [1][2][3]. Loss of Dnd1 expression in primordial germ cells results in their death during embryogenesis, thus causing sterility in adults. In 129 strain mice, inactivation of Dnd1, as in the 129-Ter mouse strain, causes loss of germ cells and in addition, development of testicular germ cell tumors [3]. Germ cell tumor development in 129-Ter/Ter (Dnd1-/-) mice is also initiated during embryonic stages [4][5][6][7].
Based on analysis using the NCBI conserved domain database (CDD), the mouse Dnd1 protein, DND1, has at least one RNA recognition motif (RRM) [3]. DND1 has been shown to bind to the 39-UTR of mRNAs to displace miRNAs that bind to adjacent sites on the same mRNA [8]. miRNA are small RNAs (20-22 nt) that regulate gene expression, mainly by post-transcriptional processes [9][10][11]. Evidence indicates that binding of DND1 to the 39-UTR of p27 or LATS2 mRNA hinders access of miR-221 or miR-372, respectively [8,12]. This results in increased p27 or LATS2 protein levels because DND1 inhibits miRNA-mediated repression of protein expression.
As DND1 is specifically expressed in germ cells, it serves to differentially regulate expression of mRNA in germ cells. During early development, germ cells are set aside from the somatic cells of the embryo on the basis of differential regulation of mRNA in germ cells compared to somatic cells [13,14]. For example, in zebrafish germ cells, Dnd1 binds to nanos1 and tdrd7 mRNA to displace miR-430 binding to mRNA. This prevents miR-430 mediated inhibition of nanos1 and tdrd7 expression in germ cells [8]. In contrast, miR-430 inhibits nanos1 and tdrd7 expression in somatic cells because somatic cells do not express Dnd1. These observations provide an explanation as to why lack of Dnd1 in germ cells causes loss of expression of germ cell specific proteins therefore resulting in germ cell death. However, it is not clear why lack of Dnd1 causes germs cells to become transformed as in the Ter mouse strain. One possibility is that absence of DND1 may cause deficiency of tumor suppressor proteins and this may cause germ cell tumor development.
Mouse DND1 protein shows highest homology to ACF (APOBEC1 complementation factor) [5,3]. The overall amino acid identity between DND1 and ACF is 34%. There is 48% identity between the RNA recognition motif (RRM) of DND1 and the first RRM of ACF (Fig. 1a). ACF is the essential RNA-binding co-factor of APOBEC1 and together they comprise the RNA editing enzyme complex (editosome) [15,16]. APOBEC1 together with ACF converts specific cytidines to uridines in the apolipoprotein B transcript and other mRNAs. On the basis of the homology between DND1 and ACF, we hypothesized that DND1, like ACF, may interact with APOBEC-like proteins.
APOBEC proteins are present in vertebrates from zebrafish to man [17]. Members of the family have zinc-binding domains with homology to bacterial cytidine deaminase. The APOBEC family members in the mouse that have RNA or DNA deaminase activity, or both are: APOBEC1, APOBEC2, APOBEC3 and AICD (activation-induced cytidine deaminase; also known as AID). The function of APOBEC2 is not known. It is expressed mainly in muscle cells [18] and has lower, but still detectable cytidine deaminase activity compared to APOBEC1. APOBEC3, is expressed in the testes, adult germ cells [19] and a wide variety of tissues. Human APOBEC3 is able to hypermutate the cDNA derived during replication of exogenous and endogenous retroviruses in cells. Thus APOBEC3 inhibits viral replication [20] and functions to maintain innate immunity of cells. AICD is found mainly in B-cells and is involved in class-switch recombination and somatic hypermutation of antibody genes [21]. Deamination within the immunoglobulin loci by AICD initiates antibody gene diversification. A new Apobec gene family member, Apobec4 was recently identified by genomic analysis but its protein and enzyme function has not been determined [22]. Mice and humans encode one gene each for APOBEC1, APOBEC2, APOBEC4 and AICD. The mouse genome also encodes one Apobec3 gene on chromosome 15. However, in humans there has been an expansion of the APOBEC3 gene family such that eight APOBEC3 genes are tandemly encoded on human chromosome 22q13.1 [17,19]. These genes are designated APOBEC3A to APOBEC3H. Of these, APOBEC3G and APOBEC3F have been extensively studied because of their ability to restrict human immunodeficiency virus (HIV) [23][24][25].
Aberrant expression of APOBECs in the cell has been proposed to cause promiscuous nucleotide modification and genomic instability [26]. Human APOBEC1 and APOBEC3 exhibit DNA mutator activity in Escherichia coli assays [27]. They caused DNA mutation through cytidine (dC) deamination and demonstrated target sequence specificity. Thus, APOBEC proteins are potential mutators and this has possible implications in cancer development. Because APOBECs have the potential ability to mutate cellular nucleic acids, their activity in cells is likely to be tightly regulated.
Recent studies on APOBEC3 indicate that it possesses cellular functions in addition to its ability to restrict retroviruses and retrotransposons. Human APOBEC3 was found to prevent microRNA-mediated inhibition of protein translation [28]. This did not require the cytidine deaminase activity of APOBEC3G. In addition, a number of reports now show that APOBEC3G interacts with a variety of RNA-binding proteins and locates to Pbodies and stress granules [29][30][31]. Very interestingly, this novel function of APOBEC3 parallels the role of DND1 in inhibiting miRNA function.
We investigated whether mouse DND1 can interact with any of the APOBEC proteins. Here, we report that DND1 can interact specifically with APOBEC3. In light of recent reports pertaining to modulation of miRNA function by both DND1 and APOBEC3, it is likely that these two proteins may act synergistically or oppose each other to modulate miRNA function.

Isolation of Apobec cDNAs from testes of 129 mice
We previously found that inactivation of Dnd1 results in sterility as well as increased testicular tumors in the 129 mouse strain [3]. The DND1 protein shows highest overall homology to ACF (APOBEC1 complementation factor) (Fig. 1a). We sought to determine if DND1, like ACF [15,16], has the ability to interact with any of the APOBEC family proteins that are normally expressed in mouse testes.
As a first step, we determined the Apobec transcripts that are present in mouse testes. We designed primers to specifically amplify Apobec transcripts from RNA derived from the testes of the 129 mouse strain. RT-PCR amplified Apobec1, Apobec2 and two isoforms of Apobec3 (Fig. 1c). Sequencing of the RT-PCR products indicated that we had isolated two isoforms of Apobec3 that differ because they contain 8 and 9 exons, respectively (Fig. 1b). The 8-exon Apobec3 isoform has an amino acid sequence identical to that of the 9-exon isoform except that it lacks 33 amino acids in the central part of the protein. The 8-exon isoform is translated from an alternately spliced mRNA that skips exon 5 of Apobec3 yet retains the same reading frame. These two isoforms of mouse Apobec3 have been previously described in the database [http://www.ensembl.org/Mus_musculus/ geneview?gene = ENSMUSG00000009585].
Although Apobec1 and Apobec2 have not previously been reported to be present in the testes, we were able to detect their transcripts by RT-PCR indicating that low levels of these Apobecs are in fact present in the testes. We were unable to isolate the AICD transcript from testes RNA possibly because its expression is restricted to Blymphocytes. The full-length cDNAs of the Apobec genes were cloned into pBK-CMV vectors (Stratagene), sequence-verified and used as templates for in vitro transcription and translation reactions to generate [ 35 S]methionine-labeled APOBEC proteins.

DND1 interacts with APOBEC proteins in vitro
The TnT Coupled Reticulocyte Lysate Systems (Promega) was used to generate [ 35 S]methionine-labeled APOBEC proteins in in vitro transcription and coupled translation reactions (see Experimental Procedures section) (Fig. 2b). For controls, human APOBEC1 (hA1) and human ACF were also generated by in vitro transcription/translation of cloned expression constructs (Fig. 2e). The sizes and amount of translated [ 35 S]methionine-labeled APOBEC protein products were determined by electrophoreses.
To test whether any of the APOBEC family members can interact with DND1 in vitro, [ 35 S]methionine-labeled APOBEC proteins were incubated with purified GST(glutathione S-transferase)-DND1 [32] (Fig. 2a). Glutathione (GST) Sepharose 4B beads were then added to the mix. In control reactions, equal amounts of [ 35 S]methionine-labeled APOBEC proteins were incubated with beads only. The glutathione Sepharose 4B beads bind to the GST moiety of GST-fusion proteins (in this case, GST-DND1) and should therefore ''pull-down'' proteins that associate with GST-DND1. At the end of the incubation period, the Sepharose 4B beads were pelleted and washed to reduce nonspecific binding. The proteins bound to the beads were released into loading dye by heating, prior to electrophoreses. In a second control, [ 35 S]methionine-labeled APOBEC proteins were incubated with purified GST protein and beads ( Fig. 2a and f).
These results show that APOBEC3 is able to specifically bind to GST-DND1. Both isoforms of APOBEC3, the 9-exon isoform and the 8-exon isoform, showed specific binding to GST-DND1a (Fig. 2c, lanes 5-8) and GST-DND1b (Fig. 2d, lanes 3-6). The binding of APOBEC3 is specific to the DND1 moiety of GST-DND1 because APOBEC3 did not bind to GST protein ( . Mouse and human APOBEC1 were also able to bind to GST-DND1 but there was significant non-specific binding in the control lanes (Fig. 2c, d and e, lanes 1 & 2). Thus, APOBEC1 may also be able to bind to DND1 but with lower affinity compared to APOBEC3. APOBEC2 or ACF did not bind to GST-DND1 (Fig. 2e).
Further, we tested whether the in vitro binding of GST-DND1 to APOBEC3 is dependent on RNA or DNA that may be present in the in vitro transcription/translation reactions. However, treatment of [ 35 S]methionine-labeled APOBEC3-GST-DND1 complexes with excess RNase or DNase did not affect binding of the two proteins (Fig. 2g). This indicates that any RNA or DNA present in the TnT coupled transcription/translation rabbit reticulocyte lysates (Promega) was not involved in promoting binding of APOBEC3 to DND1. In summary, the data indicates that APOBEC3 is able to specifically bind to DND1. These experiments provided the first evidence that DND1 can interact directly with APOBEC3.

DND1 interacts with APOBEC3 in mammalian cells
Next, we tested whether DND1 can interact with APOBEC proteins in mammalian cells. HA-tagged Dnd1 and myc-tagged Apobec plasmid constructs were co-transfected into 293T cells. myc-APOBEC1, 2, and 3 expression in transfected 293T cells were detected by immunoblotting with anti-myc ( Fig. 3a, top panel). Expression of HA-tagged DND1 was also detected using anti-DND1 antibody, antibody C [32] (Fig. 3a, lower panel). The lysates were used for immunoprecipitation with anti-HA antibody to ''pull-down'' HA-DND1 and associated proteins. No antibody was added to controls. After immunoprecipitation with anti-HA antibody, electrophoresis and transfer to membranes, western blotting was performed using anti-myc antibody.
These results are evidence that DND1 can associate with APOBEC3 in mammalian cells. This in vivo pull-down of tagged proteins is a second technique used to validate the previous in vitro binding results. Interaction of APOBEC3 to GST-DND1 in vitro is likely because of direct binding of APOBEC3 to GST-DND1 whereas ''pull-down'' of myc-APOBEC3 by HA-DND1 from cell lysates suggests interaction of the proteins but which may not necessarily be due to direct binding. Additional co-factors present in mammalian cells could bridge the interactions between the HA-DND1 and myc-APOBEC3 proteins. To summarize, the in vivo pull down experiment in 293T cells demonstrates that DND1 and APOBEC3 are present in the same protein complexes in mammalian cells.

DND1 ''pulls-down'' endogenous APOBEC3 from mouse testes
We next tested whether DND1 interacts with APOBEC3 in mouse tissues. Both DND1 and APOBEC3 are expressed in adult testes [32,33,3]. Because antibodies for immunoprecipitation of DND1 are currently not available, we used purified GST-DND1 to perform in vivo ''pull-down'' experiments. We incubated GST-DND1 protein with mouse testes lysate. The GST-DND1 was then incubated with Sepharose 4B beads to pull down GST-DND1 and any associated proteins. The beads were subsequently washed, and then boiled in loading dye prior to electrophoresis and transfer to membranes. To detect APOBEC3, immunoblotting was performed with polyclonal, rabbit anti-mouse APOBEC3 antibody (Upstate). This antibody detects an epitope in the Nterminus of mouse APOBEC3.
Our results showed that GST-DND1a was able to ''pull-down'' APOBEC3 from mouse testes (Fig. 4a). APOBEC3 from mouse testis was the same size as myc-APOBEC3 expressed in cells transfected with myc-Apobec3 expression vector (Fig. 4a). In this experiment, GST-DND1b was not able to efficiently ''pull-down'' APOBEC3 from mouse testes and this suggests that DND1a isoform interacts more efficiently with endogenous, testes APOBEC3. DND1a isoform is expressed in germ cells during embryogenesis as well as in adult stages. In contrast, DND1b is expressed in meiotic and postmeiotic germ cells in adult mice [32].

Apobec3 transcripts in mouse germ cells and developing gonads
Germ cell tumors arise in the developing gonads (genital ridges) of the male embryo. Dnd1 transcripts are detected in germ cells soon after germ cell specification [34] and in mouse embryonic gonads (genital ridges) [32,3]. Dnd1 encoded protein, DND1, is also expressed in embryonic germ (EG) cells grown in culture   [32,3]. EG cells are totipotent cells and have been isolated from primordial germ cells of embryos of embryonic (E) 8.5 -E 11.5 stages from hybrid strains of B6C3 F1 mice [35,36]. Although expression of Apobec3 in adult tissues has been described [37], the expression of Apobec3 in embryonic stages has not been examined. We therefore examined the expression of both Dnd1 and Apobec3 transcripts in embryonic germ (EG) cells and in genital ridges (embryonic gonads).
RT-PCR indicated the presence of Apobec3 and Dnd1 transcripts in EG cells (Fig. 4b). Apobec3 and Dnd1 were not detected in RNA from mouse embryo fibroblasts (MEFs), which are the feeder cell layers on which EG cells grow (Fig. 4c).
In addition, we also isolated embryonic gonads (genital ridges) from 129 strain, male E13.5 embryos. RT-PCR for Apobec3 and Dnd1 transcripts was carried out on total RNA prepared from the E13.5 genital ridges. Dnd1 and Apobec3 transcripts are expressed in E13.5 genital ridges (Fig. 4b). Interestingly, RT-PCR indicated that EG cells express higher levels of the shorter, 8-exon isoform of Apobec3 compared to that in the genital ridges. One reason for this could be because the EG cells are derived from a different mouse strain, B6C3 hybrid, whereas the genital ridges are from 129. In conclusion, we demonstrate that both Apobec3 and Dnd1 are expressed in embryonic gonads and in embryonic germ cells of the mouse.

DND1 and APOBEC3 sequester to peri-nuclear sites
To visualize the cellular localization of the DND1 and APOBEC3, we transfected expression plasmids encoding GFP-Dnd1 and mCherry-Apobec3 into different mammalian cells such as COS7, NIH3T3 and 293T cells. The transfected cells were observed using a Zeiss LSM 510 confocal microscope. Overall, when either GFP-DND1 or mCherry-APOBEC3 were transfected in cells, the expression of the fluorescent-tagged proteins was predominantly in the cytoplasm (Fig. 5a and d). However, when both GFP-DND1 and mCherry-APOBEC3 were co-transfected in COS7 cells, we observed a sharp overlap of the green and red signals to form a ring-like zone surrounding the nucleus indicating co-localization of the GFP-DND1 and mCherry-APOBEC3 fluorescent signals to perinuclear sites (Fig. 5b, c and f). The colocalization suggests that DND1 and APOBEC3 likely sequester each other. In contrast to results in COS7 cells, although both GFP-DND1 and mCherry-APOBEC3 were expressed in the cell cytoplasm, the sequestration of GFP-DND1 and mCherry-APOBEC3 to perinuclear regions was somewhat less obvious in NIH3T3 cells (Fig. 6a,  b and d) and less so in 293T cells (Fig. 6 e, g and f). One explanation as to why we observed co-localization and sequestration in COS7 but not in 293T cells could be because COS7 cells may harbor cellular factors that facilitate sequestration of GFP-DND1 and mCherry-APOBEC3. The confocal microscopy results of DND1 and APOBEC3 localization were identical when GFP or mCherry were cloned either as N-terminus or C-terminal fusion proteins to DND1 or APOBEC3, respectively (data not shown).
Human GFP-APOBEC3 orthologs (APOBEC3D, APOBEC3F and APOBEC3G) have been reported to be expressed in the cytoplasm [38,33] similar to the distribution that we observe for mouse APOBEC3. More detailed studies show that human APOBEC3 localizes to cytoplasmic bodies P-bodies and stress granules [29,31]. Future work will examine if both DND1 and APOBEC3 also localize to P-bodies in cells.
In our transfection experiments using fluorescently-tagged APOBEC3, we did not observe localization of mCherry-APOBEC3 to discreet cytoplasmic bodies but to be generally present in the cytoplasm (Fig. 5d). Thus, our results are somewhat different from reports of APOBEC3 localization to discreet cytoplasmic bodies [29]. This difference in observations could be due to variability in cell lines, transfection conditions or because mouse APOBEC3 was used in our studies as opposed to human APOBEC3G in the other studies.

APOBEC3 interacts with DND1
We have used several experimental methods to demonstrate that DND1 interacts with APOBEC3 in vitro as well as in vivo in mammalian cells and tissues. First, APOBEC3, generated in vitro, interacts specifically with DND1. We ruled out the presence of contaminating RNA or DNA in the in vitro translation mix that facilitates DND1-APOBEC3 interaction. We observed that mouse and human APOBEC1 were also able to bind to DND1 but there was significant non-specific binding of APOBEC1 to the Sepharose 4B beads as well. This suggests that APOBEC1 may also interact with DND1, although the interaction may be weaker and with lower affinity.
Second, DND1 pulled-down APOBEC3 from mammalian cells when tagged proteins were generated in transfected cells. Thus, DND1 and APOBEC3 are found in the same protein complex in mammalian cells. Recent reports indicate that human APO-BEC3G interacts with a number of cellular proteins with RNA binding domains [29,31]. In most cases, direct interaction between APOBEC3G and the proteins did not appear to be the case and the interaction between the proteins was likely through some shared mRNA. In other cases, for example, interaction of APOBEC3 with the proteins, Argonaute-1 and Argonaute-2, appeared not to be facilitated by RNA because the interaction was RNAse-resistant [29]. In the same study, 293T cells transfected with tagged-APOBEC3G resulted in the 'pull-down' and identification of a number of other cellular protein(s) [29] many of which were RNA binding proteins. Among the other proteins that were 'pulled-down' together with APOBEC3G was the HuR protein.
HuR is an AU-rich element binding protein which has been shown to bind to the 39-UTR of CAT-1 mRNA to prohibit access to miR-122 [39]. Thus, HuR is another RNA binding protein with a functional role similar to DND1.
Third, our studies demonstrate that GST-DND1 pulls-down endogenous APOBEC3 from mouse testes. In this case, pull-down of the endogenous, mouse testes APOBEC3 was more effective by DND1a compared to DND1b. This was unlike the interaction of in vitro translated APOBEC3 with GST-DND1a and GST-DND1b, where both isoforms were equally able to interact with APOBEC3. One explanation for this is that testes APOBEC3 may be post-translationally modified such that it influences its interaction with DND1a or DND1b.
Fourth, fluorescent-protein tagged DND1 and APOBEC3 proteins sequestered near peri-nuclear sites in COS7 cells. This phenomenon appears to be cell specific and was observed in COS7 cells but not in some other cell types. An explanation for this may be that additional cellular factors present in COS7 are responsible for mediating sequestration of DND1 and APOBEC3. The pull-down experiments of DND1 with APOBEC3 do not unambiguously indicate a direct interaction of the two proteins. However, taking into consideration the cell-type specific sequestration of fluorescently tagged DND1 and APOBEC3, it suggests that the interaction of DND1 and APOBEC3 may likely be mediated by other factors in the cell.
Studies by three independent groups report the localization of human APOBEC3G to P-bodies and stress granules in 293T cells Figure 6. Localization of APOBEC3 and DND1 in NIH3T3 and 293T cells. (a-d) Co-transfection of mCherry-Apobec3 and GFP-DND1 expression constructs into NIH3T3 cells. Cells were fixed with 2% paraformaldehyde before visualization of fluorescent signal by confocal microscopy. (a) Red fluorescence due to mCherry-APOBEC3 was imaged using red filter (excitation at 548 nm); (b) green fluorescence due to GFP-DND1 was imaged using green filter (excitation at 488 nm); (d) merged image of red and green filters showing APOBEC3 and DND1 co-localize (yellow signal) near perinuclear region in NIH3T3 cells. (c) The DIC (differential interference contrast) image of the cells. (e-g) Co-transfection of mCherry-Apobec3 and GFP-DND1 expression constructs into 293T cells. Cells were fixed with 2% paraformaldehyde before visualization of fluorescent signal by confocal microscopy. (e) Image of red fluorescence due to mCherry-APOBEC3 (red filter; excitation at 548 nm); (g) green fluorescence due to GFP-DND1 (green filter, excitation at 488 nm) and (f) merged image of red and green filters showing APOBEC3 and DND1 co-localize to cytoplasm in 293T cells. doi:10.1371/journal.pone.0002315.g006 [29][30][31]. The evidence suggests that the cytidine deaminase activity of APOBEC3G is likely inhibited in these cytoplasmic granules [40,28,30]. In light of this, the consequence of DND1-APOBEC3 interaction for either APOBEC3 or DND1 function remains to be elucidated.
In addition to the experimental demonstration of DND1-APOBEC3 interaction, we found that both Dnd1 and Apobec3 transcripts are detected in germ cells and in the developing embryonic gonads. The transcripts are present in germ cells and in genital ridges during embryonic stages when DND1 function is required for germ cell viability. Moreover, lack of Dnd1 at these embryonic stages also results in initiation of germ cell tumors in the 129 strain male [5,7].
Although cytidine deamination appears to be the primary mechanism by which APOBEC3 inhibits retrovirus replication [43,38,46,50], there is also a large body of evidence suggesting some novel, yet undefined, deaminase independent mechanism for the antiviral function of APOBEC3 [54][55][56]33]. Mouse APO-BEC3 has also been shown to have anti-retroviral activity [20]. Mouse APOBEC3 has two cytidine deaminase domains (CDD) [57,19]. The proximal CDD is involved in deamination whereas the distal CDD is involved in dimerization of APOBEC3 proteins and for viral encapsidation [57].
Recent reports indicate additional cellular functions of APO-BEC3. APOBEC3 is able to inhibit miRNA-mediated repression of mRNA. APOBEC3 appears to allow mRNA to move from Pgranules to polysomes [28].
The role of APOBEC3 in suppressing miRNA mediated mRNA repression parallels recent reports on the role of DND1 in suppressing miRNA-mediated mRNA repression [8]. It is not clear if DND1 sequesters mRNA to prevent its access to miRNA or whether DND1 physically displaces miRNA RISC complex from mRNA [8,12]. In this respect, it will be important to examine if DND1 and APOBEC3 function synergistically or antagonistically to modulate miRNA function.
The inactivating mutation in the Dnd1 gene in Ter mice causes either sterility or testicular germ cell tumor development. We report here that DND1 interacts with APOBEC3. The functional consequences of DND1 interaction with APOBEC3 remains to be established. One possibility is that DND1 may modulate some specific antiviral or anti-retrotransposion activity of APOBEC3. DND1 may be a cellular factor that is necessary for regulating some aspect of APOBEC3 activity, especially in germ cells.
Another possibility is that DND1 and APOBEC3 function synergistically or antagonistically to modulate miRNA mediated repression of mRNA. The 39-UTR of mRNAs generally contains multiple miRNA binding sites as well as binding sites for different RNA binding proteins. In light of the reports that both DND1 and APOBEC3 bind to mRNA to inhibit miRNA mediated repression, and taking into consideration our observation of DND1-APOBEC3 interaction, our studies implicate a possible role of DND1-APOBEC3 interaction in modulating miRNA-mediated mRNA repression. Understanding the role of DND1-APOBEC3 interaction will not only shed light on the development of testicular germ cell tumors in mice and humans but may also have profound implications for our understanding of the mechanism of how cancers in general originate in humans.

In vitro transcription and translation
The full-length cDNA of the Apobecs were cloned into pBK-CMV, sequence verified and used as templates in in vitro transcription/translation reactions.

Generation and purification of GST-DND1
The cDNA of the two isoforms of mouse DND1 (isoforms a and b) were cloned in-frame into pGEX-2TK (amersham pharmacia biotech) [32]. The GST(glutathione S-transferase)-DND1 fusion proteins were induced by IPTG (isopropyl-b-D-thiogalactoside), extracted from bacteria by sonication and affinity purified using Glutathione Sepharose 4B beads. GST proteins were also generated in a similar manner for use in control reactions.

In vitro interaction reactions
Equal amounts of Apobec cDNA cloned into pBK-CMV (1 mg) were used as templates in in vitro transcription/translation reactions (TnT Coupled Reticulocyte Lysate Systems, Promega) to generate [ 35 S]methionine-labeled APOBEC proteins in a total volume of 100 mL. The amount of each [ 35 S]methionine-labeled APOBEC protein generated in the reaction was determined by electrophoreses of 5 mL on a gel.
Equal amounts (40 mL) of each of the [ 35 S]methionine-labeled APOBEC proteins were placed into two tubes. One tube of the [ 35 S]methionine-labeled APOBEC proteins was incubated with purified GST-DND1 and Glutathione (GST) Sepharose 4B beads. The second tube (control) was incubated with beads only. Glutathione Sepharose 4B binds to GST and GST-fusion proteins (in this case, GST-DND1) and should therefore ''pull-down'' proteins that associate with GST-DND1. At the end of the incubation period, the tubes were centrifuged to pellet the Sepharose 4B beads. The beads were washed to reduce nonspecific binding. Loading dye was added to the beads before heating to 95 0 C to release the proteins from the beads into the loading dye. Equal volumes of the loading dye were electrophoresed on NuPAGE-Mes gels (Invitrogen) to assess the binding of the radiolabelled APOBEC proteins to GST-DND1.
To test whether the in vitro binding of GST-DND1 to APOBEC3 is dependent on RNA or DNA that may be present in the in vitro transcription/translation reactions, [ 35 S]labeled APOBEC-3-GST-DND1 complexes were treated for 1 h with 0.08 U RNase (RNAse, DNase-free from bovine pancreas, Roche) or 5 U DNase (DNAse 1, RNase-free from bovine pancreas).

Pull-down of APOBEC3 with DND1 in mammalian cells
Dnd1 and Apobec cDNAs were cloned in frame into mammalian expression vectors (pCMV-HA or pCMV-Myc, Clontech) to generate HA(hemagglutinin) epitope-tagged DND1 or myc-tagged APOBEC proteins. Both HA-Dnd1 and myc-Apobec plasmid constructs were co-transfected into human embryonic kidney (HEK) 293T cells. The transfected 293T cells were lysed after 48 h, and immunoprecipitation using anti-HA antibody (abm) was performed to ''pull-down'' HA-DND1 and associated proteins. After electrophoresis and transfer to membranes, western blotting was performed using anti-myc antibody (Invitrogen). Control aliquots of cell lysates were not incubated with antibody.

RNA isolation from EG cells and genital ridges
EG cells were isolated from hybrid strains of B6C3 F1 mice [35]. The cells were grown on mitomycin C-treated mouse embryo fibroblasts. While in culture, some EG cell plates were periodically stained with alkaline phosphatase chromogen (abcam) to ensure they had not differentiated.
RNA isolation from male genital ridges from 129 mouse embryos at E13.5 stages was as described [32].

Fluorescent protein tagged-APOBEC3 and DND1
Mouse Dnd1 was cloned in frame and fused to fluorescent GFP (green fluorescent protein) either in the N or C-terminus in pEGFp-N3 (Clontech) and pEGFP-C1 (Clontech) vectors. Mouse Apobec3 (9-exon isoform) was cloned in frame and fused to monomeric cherry fluorescent protein (mCherry) in the pRSET-B mCherry vector [58]. The cherry fluorescent protein was substituted into the ECFP sites in the pECFP-C1 (Clontech) and pECFP-N1 (Clontech) vectors so the multiple cloning sites of C1 and N1 could be used to clone in and generate C and N-terminal cherry fusion products.
The GFP-Dnd1 and mCherry-Apobec-3 were transfected separately into 293T or COS7 cells. After 20 hours, transfected cells were fixed with 2% paraformaldehyde before visualization of fluorescent signal by Zeiss LSM 510 Confocal Microscope. Green fluorescence due to GFP-DND1 was imaged using green filter (excitation at 488 nm); red fluorescence due to mCherry-APOBEC-3 was imaged using red filter (excitation at 548 nm).